1. Field of the Invention
This invention relates to micro-discharge optical source apparatus and methods and systems for analyzing a sample.
2. Background Art
The following references are referenced herein:    [1] H. Zhu et al., “High-Sensitivity Capillary Electrophoresis of Double-Stranded DNA Fragments Using Monomeric and Dimeric Fluorescent Intercalating Dyes,” ANALYTICAL CHEMISTRY, 66, pp. 1941–8, 1994.    [2] R. P. Haugland, HANDBOOK OF FLUORESCENCE PROBES AND RESEARCH CHEMICALS, Molecular Probes, Eugene, OR, 1996.    [3] S. V. Konev, FLUORESCENCE AND PHOSPHORESCENCE OF PROTEINS AND NUCLEIC ACIDS, Plenum Press, 1967.    [4] A. S. Ladokhin, “Fluorescence Spectroscopy in Peptide and Protein Analysis,” ENCYCLOPEDIA OF ANALYTICAL CHEMISTRY, Ed. R. A. Meyers, John Wiley & Sons Ltd., 2000, pp. 5762–5779.    [5] G. D. Fasman, HANDBOOK OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, PROTEINS I, CRC Press, 1976, pp. 183–203.    [6] R. Chen, “Measurements of Absolute Values in Biochemical Fluorescence Spectroscopy,” J. RESEARCH NATIONAL BUREAU STANDARDS, 76A(6), 1972, pp. 593–606.    [7] H. Chou et al., “A Microfabricated Device for Sizing & Sorting DNA Molecules,” PROC. NATL. ACAD. SCI., 96, pp. 11–13, 1999.    [8] J. Webster et al., “Monolithic Capillary Electrophoresis Device With Integrated Fluorescence Detector,” ANAL. CHEM., 73, pp. 1622–6, 2001.    [9] M. Warren et al., “Integrated Micro-optical Fluorescence Detection System for Microfluidic Electro-Chromatography,” PROC. SPIE, v. 3878, pp. 185–192, 1999.    [10] E. Thrush et al., “Integrated Semiconductor Fluorescent Detection System for Biochip & Biomedical Applications,” PROC. SPIE, 4626, pp. 289–96, 2002.    [11] L. Que et al., “A Water Spectroscopy Microsystem with Integrated Discharge Source, Dispersion Optics, and Sample Delivery,” PROC., IEEE TRANSDUCERS CONF., Boston, June 2003.    [12] L. Que et al., “Dye-Fluorescence LEd-SpEC: A Battery-Operated, On-Chip, Wavelength-Tunable Optical Source for Detection of Biochemicals,” PROC. OF THE MICRO TOTAL ANALYSIS SYSTEMS SYMPOSIUM, Squaw Vally, Calif., October 2003.    [13] L-Tryptophan, CAS Number 73-22-3, Fischer Scientific, Catalog Number, BP395-100, Lot Number 018929.    [14] Model number K30-635 and K43-456, Edmund Industrial Optics, Inc., Barrington, N.J.    [15] L. H. Light et al., “Transistor D.C. Convertors,” PROC. OF IEEE, B, 102, pp. 775–786, 1955.
Fluorescence detection is a widely used technique for medical diagnostics and biochemical analysis. The molecules of interest fluoresce at characteristic emission wavelengths when they are illuminated at characteristic excitation wavelengths, which are shorter (and hence more energetic).
In one diagnostic approach, a fluorescent dye is used to chemically label the quantity of interest. For DNA detection, dyes which intercalate into the double-helix provide very high sensitivity and make it possible to detect attomoles of DNA base-pairs [1]. A contributing factor to high sensitivity is quantum efficiency, which is the ratio of the number of photons emitted to those absorbed in the excitation wavelengths. For example, SYBR Green I gel stain is a cyanine dye that has a quantum efficiency of 0.8 [2]. When bound to dsDNA, it is most efficiently excited by radiation over 491–503 nm, and has a broad emission spectrum over 510–600 nm, with a peak at 522 nm.
While dyes offer many attractive features, their use is not always favored or even possible. For example, proteins can be fluorescent even without the presence of a dye, and changes in this intrinsic or direct fluorescence can be indicative of structural transformations [3,4]. The intrinsic fluorescence of proteins and peptides is due to the presence of tryptophan, tyrosine or phenylalanine, which are amino acids. In contrast to the excitation and emission wavelengths for the SYBR green dye, which are in the visible portion of the spectrum, these three have absorption peaks over 250–290 nm and emission peaks over 280–350 nm, all in the deep ultra-violet (UV) region. The characteristics for tryptophan are shown in FIG. 1 [5,6]. (Note that the wavelength at which this fluorescence peaks is highly sensitive to the microenvironment, and hence it is widely used for studying protein structure and dynamics). It is noteworthy that the quantum efficiencies of these amino acids are relatively low. For example, tryptophan, which tends to dominate in fluorescence over the other two, has a quantum efficiency of only 0.19 when dissolved in water as a free amino acid [3]. These characteristic can make it relatively challenging to observe direct fluorescence.
In a typical fluorescence imaging system, the radiation source is often broad-band, so a low-pass filter is located between the source and the sample to reduce its illumination by the longer wavelengths. In addition, a high-pass filter located between the sample and the detector so as to restrict the measured signal to the fluorescence wavelengths and minimize the radiation from the source that might inadvertently leak through.
In recent years, significant research has been devoted to miniaturization of biochemical instrumentation, leading to micro-total analysis systems (also referred to as “lab-on-a-chip”). With respect to fluorescence detectors, the efforts have focused on solid-state sources such as light-emitting diodes (LEDs) and lasers (VCSELs) [7–10]. However, making these sources for deep UV wavelengths and integrating them with microfluidic systems is a major challenge.
U.S. Pat. No. 6,686,998 discloses a glow discharge apparatus having liquid electrodes including a substrate with a top surface on which cathode and anode electrodes are formed. The cathode electrode may be formed with a cathode terminal port formed to hold a liquid which is spaced from the anode electrode by an inter-electrode surface of the substrate. Electrical conductors are connected to the anode and cathode electrodes to allow a voltage to be applied between them, resulting in a glow discharge in the gap over the inter-electrode surface that causes sputtering of the liquid in the cathode terminal port into the glow discharge. Excitation by the glow discharge of the sputtered or evaporated liquid allows spectroscopic analysis of the constituents of the liquid in the electrode. The glow discharge apparatus utilizes liquid electrodes which allow spectrometric analysis of liquid samples and particularly water samples for determining contaminants in the water. The apparatus may also be utilized as a micro light source that provides light output at visible or non-visible wavelengths that can be selected by selection of the liquid utilized in the electrodes of the materials dissolved or suspended in the electrode liquids. The device to be utilized for on-chip UV sources. Other constituents of the water in the cathode reservoir can be chosen to obtain emission at other wavelengths, including visible wavelengths.